leupeptins and Cell-Transformation--Neoplastic

During tumorigenesis, oncogene activation and metabolism rewiring are interconnected. Activated c‑

Topics: Asparagine; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; Gene Expression Regulation, Neoplastic; Glutamate-Ammonia Ligase; Glutamine; Humans; Leupeptins; Methionine Sulfoximine; Proteasome Endopeptidase Complex; Protein Isoforms; Proteolysis; Proto-Oncogene Proteins c-myc

β-catenin is a major transcriptional activator of the canonical Wnt/β-catenin signaling pathway. It is important for a series of biological processes including tissue homeostasis, and embryonic development and is involved in various human diseases. Elevated oncogenic activity of β-catenin is frequently observed in cancers, which contributes to survival, metastasis and chemo-resistance of cancer cells. However, the mechanism of β-catenin overexpression in cancers is not well defined. Here we demonstrate that the deubiquitination enzyme USP20 is a new regulator of the Wnt/β-catenin signaling pathway. Mechanistically, USP20 regulates the deubiquitination of β-catenin to control its stability, thereby inducing proliferation, invasion and migration of cancer cells. High expression of USP20 correlates with increased β-catenin protein level in multiple cancer cell lines and patient samples. Moreover, knockdown of USP20 increases β-catenin polyubiquitination, which enhances β-catenin turnover and cell sensitivity to chemotherapy. Collectively, our results establish the USP20-β-catenin axis as a critical regulatory mechanism of canonical Wnt/β-catenin signaling pathway with an important role in tumorigenesis and chemo response in human cancers.

Topics: Animals; Antineoplastic Agents; beta Catenin; Cell Line, Tumor; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Female; HEK293 Cells; Humans; Leupeptins; Mice; Mice, Nude; Neoplasms; RNA Interference; RNA, Small Interfering; Ubiquitin Thiolesterase; Ubiquitination; Wnt Signaling Pathway

The insulin-like growth factor-1 system, including its critical mediator insulin receptor substrate-1 (IRS-1), is involved in regulating osteosarcoma (OS) cell proliferation or differentiation. The aim of this study is to define the role of IRS-1 in OS cells by assessing the contribution of IRS-1 in the differentiation of human and murine OS cell lines and mouse mesenchymal stem cells (MSCs) and found that the basal level of IRS-1 is important for the initiation of differentiation. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. In vivo studies showed that OS cells over-expressing IRS-1 have increased metastatic potential and tumor growth. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that inhibited osteoblastic differentiation, suggesting a role for proteasomal regulation in maintaining the appropriate expression level of IRS-1. Thus, precise regulation of IRS-1 expression level is critical for determining the differentiating capacity of MSCs and OS cells, and that derangement of IRS-1 levels can be a critical step in OS transformation.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Humans; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Leupeptins; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Osteoblasts; Osteocalcin; Osteosarcoma; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; RNA Interference; RNA, Small Interfering; Signal Transduction; Sp7 Transcription Factor; Transcription Factors

Pyruvate kinase M2 (PKM2) is expressed at high levels during embryonic development and tumour progression and is important for cell growth. However, it is not known whether it directly controls cell division. Here, we found that Aurora B phosphorylates PKM2, but not PKM1, at T45; this phosphorylation is required for PKM2's localization and interaction with myosin light chain 2 (MLC2) in the contractile ring region of mitotic cells during cytokinesis. PKM2 phosphorylates MLC2 at Y118, which primes the binding of ROCK2 to MLC2 and subsequent ROCK2-dependent MLC2 S15 phosphorylation. PKM2-regulated MLC2 phosphorylation, which is greatly enhanced by EGF stimulation or EGFRvIII, K-Ras G12V and B-Raf V600E mutant expression, plays a pivotal role in cytokinesis, cell proliferation and brain tumour development. These findings underscore the instrumental function of PKM2 in oncogenic EGFR-, K-Ras- and B-Raf-regulated cytokinesis and tumorigenesis.

Topics: Amides; Animals; Aurora Kinase B; Brain Neoplasms; Cardiac Myosins; Carrier Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cytokinesis; ErbB Receptors; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Leupeptins; Membrane Proteins; Mice; Mice, Nude; Mitosis; Myosin Light Chains; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Pyridines; Pyruvate Kinase; ras Proteins; rho-Associated Kinases; RNA Interference; RNA, Small Interfering; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Intrinsic stress response pathways are frequently mobilized within tumor cells. The mediators of these adaptive mechanisms and how they contribute to carcinogenesis remain poorly understood. A striking example is heat shock factor 1 (HSF1), master transcriptional regulator of the heat shock response. Surprisingly, we found that loss of the tumor suppressor gene neurofibromatosis type 1 (Nf1) increased HSF1 levels and triggered its activation in mouse embryonic fibroblasts. As a consequence, Nf1-/- cells acquired tolerance to proteotoxic stress. This activation of HSF1 depended on dysregulated MAPK signaling. HSF1, in turn, supported MAPK signaling. In mice, Hsf1 deficiency impeded NF1-associated carcinogenesis by attenuating oncogenic RAS/MAPK signaling. In cell lines from human malignant peripheral nerve sheath tumors (MPNSTs) driven by NF1 loss, HSF1 was overexpressed and activated, which was required for tumor cell viability. In surgical resections of human MPNSTs, HSF1 was overexpressed, translocated to the nucleus, and phosphorylated. These findings reveal a surprising biological consequence of NF1 deficiency: activation of HSF1 and ensuing addiction to this master regulator of the heat shock response. The loss of NF1 function engages an evolutionarily conserved cellular survival mechanism that ultimately impairs survival of the whole organism by facilitating carcinogenesis.

Topics: Active Transport, Cell Nucleus; Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Neurofibromatosis 1; Heat Shock Transcription Factors; Hot Temperature; Humans; Leupeptins; Macrolides; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Proteins; Nerve Sheath Neoplasms; Neurofibromin 1; NIH 3T3 Cells; Phosphorylation; Protein Processing, Post-Translational; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Transcription Factors; Withanolides

Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H(2)O(2)) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H(2)O(2) impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-L-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.

Topics: Acetylcysteine; Animals; Blast Crisis; Carrier Proteins; Cell Proliferation; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; Free Radical Scavengers; Gene Expression Regulation, Leukemic; GTP-Binding Proteins; HL-60 Cells; Humans; Hydrogen Peroxide; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leupeptins; Lymphoma; Mice; Mice, Transgenic; Mutagenesis, Site-Directed; Neoplasms, Experimental; Nuclear Proteins; Oxidants; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Multimerization; Proto-Oncogene Proteins c-myc; RNA-Binding Proteins

A stem cell model of prostate cancer tumourigenesis explains progression to castration resistant prostate cancer (CRPC) and offers novel perspectives in targeting this cancer in its more advanced forms. Androgen receptor (AR) regulated pathways are central mechanisms in progression to CRPC. However, AR was thought to be lacking in prostate stem cell enriched fractions. Potential low levels of AR expression in stem cell enriched cells were investigated and potential direct effects of androgen were examined.. Human prostate stem cell enriched populations, based on high α(2)β(1) integrin expression (α(2)β(1)(hi)), were selected from primary human prostate tissue in men undergoing transurethral prostatectomy or cystoprostatectomy. Effects on differentiation were assayed with flow cytometry using differentiation-specific markers.. Low levels of AR were demonstrable in α(2)β(1)(hi) cells following inhibition of the proteasome using MG132. Furthermore, a direct effect of androgen was shown in stabilising/inducing AR expression. Androgen treatment of α(2)β(1)(hi) cells was associated with the induction of differentiation using a number of differentiation-specific markers (prostatic acid phosphatase, cytokeratin 18 and AR) with increases ranging from 49% to 67% (p<0.05). These effects were blocked with the AR-specific inhibitor bicalutamide (p<0.05). These data support a role of direct androgen activity on stem cell enriched cells in the prostate and the implications of these findings are discussed.

Topics: Aged; Aged, 80 and over; Androgen Antagonists; Androgen Receptor Antagonists; Androgens; Anilides; Antigens, Differentiation; Biomarkers, Tumor; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; Epithelial Cells; Humans; Leupeptins; Male; Metribolone; Middle Aged; Neoplastic Stem Cells; Nitriles; Prostatic Neoplasms; Receptors, Androgen; Testosterone Congeners; Tosyl Compounds

PPP1R13L was initially identified as a protein that binds to the NF-kappaB subunit p65/RelA and inhibits its transcriptional activity. It also binds p53 and inhibits its action. One set of experimental findings based on overexpression of PPP1R13L indicates that PPP1R13L blocks apoptosis. Another set of experiments, based on endogenous production of PPP1R13L, suggests that the protein may sometimes be pro-apoptotic. We have used primary mouse embryonic fibroblasts (MEFs), dually transformed by HRAS and adenovirus E1A and differing in their p53 status, to explore the effects of PPP1R13L overexpression, thus examining the ability of PPP1R13L to act as an oncoprotein. We found that overexpression of PPP1R13L strongly accelerated tumor formation by RAS/E1A. PPP1R13L overexpressing cells were depleted for both p53 and active p65/RelA and we found that both p53-dependent and -independent apoptosis pathways were modulated by PPP1R13L. Finally, studies with the proteasome inhibitor MG132 revealed that overexpression of PPP1R13L causes faster p53 degradation, a likely explanation for the depletion of p53. Taken together, our results show that increased levels of PPP1R13L can increase tumorigenesis and furthermore suggest that PPP1R13L can influence metastasis.

Topics: Adenovirus E1A Proteins; Animals; Apoptosis; Cell Cycle; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Embryo, Mammalian; Fibroblasts; Intracellular Signaling Peptides and Proteins; Leupeptins; Mice; Mice, Knockout; Mice, Nude; Neoplasm Invasiveness; Neoplasms, Experimental; ras Proteins; Repressor Proteins; Signal Transduction; Transcription Factor RelA; Transfection; Tumor Suppressor Protein p53

The N-alpha-acetyltransferase NatB, composed in Saccharomyces cerevisiae by the Nat3p and Mdm20p subunits, is an important factor for yeast growth and resistance to several stress agents. However, the expression and functional role of the mammalian counterpart has not yet been analysed. Here, we report the identification of Nat3p human homologue (hNAT5/hNAT3) and the characterization of its biological function. We found that hNAT5/hNAT3 silencing in HeLa cells results in inhibition of cell proliferation and increased sensitivity to the pro-apoptotic agent MG132. Moreover, inhibition of hNAT5/hNAT3 expression induces p53 activation and upregulation of the antiproliferative protein p21(WAF1/CIP1). The changes of the cellular transcriptome after hNAT5/hNAT3 knockdown confirmed the involvement of this protein in cell growth and survival processes. Among the genes differentially expressed, we observed upregulation of several p53-dependent antiproliferative and pro-apoptotic genes. In the c-myc transgenic mice, which is a model of inducible hepatocarcinoma, we found that hNAT5/hNAT3 was upregulated when the tumour was induced. In accordance with this observation, we noticed increased hNAT5/hNAT3 protein level in neoplastic versus non-neoplastic tissue in a high proportion of patients with hepatocellular carcinoma. Consequently, our results suggest that hNAT5/hNAT3 is required for cellular proliferation and can be implicated in tumour growth.

Topics: Acetyltransferases; Adenoviridae; Animals; Apoptosis; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Gene Silencing; HeLa Cells; Humans; Kidney; Leupeptins; Mice; Mice, Transgenic; N-Terminal Acetyltransferase A; N-Terminal Acetyltransferase E; RNA, Small Interfering; Tumor Suppressor Protein p53

To determine whether proteasome inhibition could reverse E6-mediated p53 degradation, cause selective growth inhibition, and induce apoptosis in human papillomavirus E6-transformed primary tonsil epithelial cells.. Primary human and mouse tonsil epithelial cell lines were transformed with a retrovirus containing human papillomavirus 16 oncogenes. MG132 was used to inhibit proteasome degradation in vitro and in vivo, and biochemical assays regarding p53 and apoptosis were performed.. In cells that express E6, proteasome inhibition with MG132 restored p53 protein levels and decreased proliferation in a dose-dependent fashion that was significantly more pronounced compared with controls. However, inhibition of proliferation occurred at a lower concentration than restoration of p53 protein expression. Also, wild-type and p53 knockout mouse tonsil epithelial cells that express E6 had near-identical inhibition of growth, suggesting that growth inhibition was p53 independent. In vivo studies did not demonstrate any growth inhibition.. The findings suggest that proteasome inhibition preferentially inhibits proliferation in cells expressing E6 through a p53-independent mechanism.

Topics: Animals; Apoptosis; Caspase 3; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Dose-Response Relationship, Drug; Epithelial Cells; Head and Neck Neoplasms; Humans; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Knockout; Oncogene Proteins, Viral; Palatine Tonsil; Protease Inhibitors; Proteasome Inhibitors; Repressor Proteins; Tumor Suppressor Protein p53

Several food polyphenols act as chemopreventers by reducing the incidence of many types of cancer, especially in colon epithelia. In this study, we have investigated whether the flavonoid quercetin can modulate cell proliferation and survival by targeting key molecules and/or biological processes responsible for tumor cell properties. The effect of quercetin on the expression of Ras oncoproteins was specifically studied using systems of either constitutive or conditional expression of oncogenic RAS in human epithelial cells. Our findings suggest that quercetin inhibits cell viability as well as cancer cell properties like anchorage-independent growth. These findings were further supported at the molecular level, since quercetin treatment resulted in a preferential reduction of Ras protein levels in cell lines expressing oncogenic Ras proteins. Notably, in cells that only express wild-type Ras or in those where the oncogenic Ras allele was knocked out, quercetin had no evident effects upon Ras levels. We have shown that quercetin drastically reduces half-life of oncogenic Ras but has no effect when the cells are treated with a proteasome inhibitor. Moreover, in Ha-RAS-transformed cells, quercetin induces autophagic processes. Since quercetin downregulates the levels of oncogenic Ras in cancer cells, we propose that this flavonoid could act as a chemopreventive agent for cancers with frequent mutations of RAS genes.

Topics: Autophagy; Cell Proliferation; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Genes, ras; Humans; Leupeptins; Oncogene Protein p21(ras); Quercetin; ras Proteins; Tumor Cells, Cultured

Abnormal differentiation in epithelial stem cells or their immediate proliferative progeny, the transiently amplifying population (TAP), may explain malignant pathogenesis in the human prostate. These models are of particular importance as differing sensitivities to androgen among epithelial cell subpopulations during differentiation are recognised and may account for progression to androgen independent prostate cancer. Androgens are crucial in driving terminal differentiation and their indirect effects via growth factors from adjacent androgen responsive stroma are becoming better characterised. However, direct effects of androgen on immature cells in the context of a prostate stem cell model have not been investigated in detail and are studied in this work. In alpha2beta1hi stem cell enriched basal cells, androgen analogue R1881 directly promoted differentiation by the induction of differentiation-specific markers CK18, androgen receptor (AR), PSA and PAP. Furthermore, treatment with androgen down-regulated alpha2beta1 integrin expression, which is implicated in the maintenance of the immature basal cell phenotype. The alpha2beta1hi cells were previously demonstrated to lack AR expression and the direct effects of androgen were confirmed by inhibition using the anti-androgen bicalutamide. AR protein expression in alpha2beta1hi cells became detectable when its degradation was repressed by the proteosomal inhibitor MG132. Stratifying the alpha2beta1hi cells into stem (CD133(+)) and transient amplifying population (TAP) (CD133(-)) subpopulations, AR mRNA expression was found to be restricted to the CD133(-) (TAP) cells. The presence of a functional AR in the TAP, an androgen independent subpopulation for survival, may have particular clinical significance in hormone resistant prostate cancer, where both the selection of immature cells and functioning AR regulated pathways are involved.

Topics: AC133 Antigen; Acid Phosphatase; Aged; Aged, 80 and over; Androgen Antagonists; Anilides; Antigens, CD; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Epithelial Cells; Fibroblast Growth Factor 7; Glycoproteins; Humans; Integrin alpha2beta1; Keratin-18; Leupeptins; Male; Metribolone; Middle Aged; Neoplastic Stem Cells; Nitriles; Peptides; Phenotype; Prostate-Specific Antigen; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Tyrosine Phosphatases; Receptors, Androgen; RNA, Messenger; Signal Transduction; Testosterone Congeners; Tosyl Compounds

HER-2/neu proto-oncogene is overexpressed in about one fourth of human breast cancers. AP-2 transcription factors bind to the HER-2/neu gene promoter and activate its expression. In a striking concurrence, anomalous abundance of AP-2alpha protein or its homolog AP-2gamma is also detected with HER-2/neu protein in mammary tumor-derived cell lines. This suggests that the deregulation of AP-2 is the preceding pathogenic event and probably the pivotal one in this type of mammary carcinogenesis. We examined the process of AP-2alpha gene expression in mammary carcinoma cell lines to identify where the aberration had occurred. We found no amplification of the AP-2alpha gene. Its promoter was marginally upregulated; however, it did not significantly increase the mRNA levels. When the AP-2alpha protein was examined, a remarkable stability was seen in breast cancer cell lines MDA-MB-453 and SK-BR-3, with a half-life of over 30 hr. This is sharply higher than the approximate 1 hr observed in mammary epithelial cell line MCF-10A and murine cell line NIH 3T3. Treatment of MCF-10A and NIH 3T3 cells with the proteasome inhibitor MG-132 showed that AP-2alpha was ubiquitinated and its level significantly increased. Moreover, this increase was accompanied by elevated levels HER-2/neu protein. In contrast, weaker ubiquitination of AP-2alpha was seen in MDA-MB-453 and SK-BR-3 cancer cells, and MG-132 treatment did not raise the AP-2alpha level any further. These results uncover that unusual stability is the main mechanism that raises the levels of AP-2 proteins, and in addition, provide the first clue that defective ubiquitin-dependent proteasomal-degradation pathway is possibly the prime cause that affects the HER-2/neu gene and culminates in breast cancer.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; Female; Gene Amplification; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Mammary Neoplasms, Animal; Mice; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Proto-Oncogene Mas; Receptor, ErbB-2; Transcription Factor AP-2; Tumor Cells, Cultured; Ubiquitin; Up-Regulation

Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway.

Topics: Aged; Alkaloids; Amino Acid Chloromethyl Ketones; Aprotinin; Benzophenanthridines; Benzyl Compounds; Caspase 3; Caspases; Cell Division; Cell Transformation, Neoplastic; Colonic Neoplasms; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; DNA; DNA Fragmentation; Down-Regulation; Humans; Hydrocarbons, Fluorinated; Leucine; Leupeptins; Male; Pepstatins; Phenanthridines; Protein Kinase C; Pyridines; Tumor Cells, Cultured

HT-29 cells derived from a human colonic adenocarcinoma, can express a typical intestinal differentiation. Undifferentiated HT-29 cells accumulate N-linked glycoproteins substituted with unprocessed carbohydrate chains before to degrade them. Conversely, carbohydrate chains of N-linked glycoproteins are classically processed in differentiated HT-29 cells. The instability of N-linked glycoproteins in undifferentiated HT-29 cells is due to their rapid delivery from the endoplasmic reticulum to a compartment with lysosomal characteristics. This catabolitic pathway involves a bypass of the Golgi apparatus.

Topics: Adenocarcinoma; Cell Transformation, Neoplastic; Colonic Neoplasms; Drug Stability; Glycoproteins; Humans; Leupeptins; Polysaccharides; Protein Processing, Post-Translational; Tumor Cells, Cultured

We investigated the effects of three serine protease inhibitors (leupeptin, soybean trypsin inhibitor, and aprotinin) on the serum-free growth of two transformed cell lines in soft agar. Aprotinin markedly enhanced the growth of rat embryo fibroblasts that had been transformed by polyoma middle T antigen (PyMLV-REF52), while having only a slight effect on the colonial growth of SV40 transformed Balb/c 3T3 cells (SV3T3-Aga). Leupeptin and soybean trypsin inhibitor, on the other hand, significantly enhanced the growth of SV3T3-Aga cells while having little effect on PyMLV-REF52 growth. We observed no stimulatory effect of any of the protease inhibitors on serum-free monolayer growth. Under conditions of excess aprotinin, PyMLV-REF52 cells were found to be unresponsive to epidermal growth factor (EGF) at a concentration that would normally stimulate agar colony growth. However, aprotinin was not capable of supporting colony formation with transforming growth factor-beta. These results indicate that aprotinin acts primarily as a protease inhibitor in spite of its structural homology to EGF and that EGF may promote the soft agar growth of these cell lines either by inhibiting proteolysis directly or by enhancing the synthesis of a serine protease inhibitor.

Topics: Antigens, Polyomavirus Transforming; Aprotinin; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Humans; Leupeptins; Serine Proteinase Inhibitors; Trypsin Inhibitor, Kunitz Soybean

Previous studies in this laboratory have quantified the fibrinolytic activity of herpesvirus-transformed cell lines and implicated the proteolytic capacity of cloned cell lines in the formation of primary and metastatic tumours. Because of the involvement of proteases in tumourigenesis in this system, we examined the effect of various protease inhibitors (alpha-1-antitrypsin, leupeptin and alpha-2-macroglobulin), as well as hamster serum containing acute phase proteins generated in response to physiological trauma, on the fibrinolytic capacity of a herpes simplex virus type 2-transformed hamster cell line. The effects of the various inhibitors on cell growth, fibrinolysis in vitro and tumourigenesis were examined. Leupeptin, alpha-1-antitrypsin, alpha-2-macroglobulin and hamster serum containing acute phase proteins were capable of inhibiting fibrinolysis in vitro, were not toxic to the cells and their action was reversible, while rejection of a mixture of protease inhibitor and transformed cells resulted in delayed tumour development.

Topics: alpha 1-Antitrypsin; alpha-Macroglobulins; Animals; Blood Proteins; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cricetinae; Fibrinolysis; Leupeptins; Neoplasms, Experimental; Protease Inhibitors; Simplexvirus

Plasminogen activator (PA) activity was analyzed in normal and transformed 10T1/2 mouse fibroblasts treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the protease inhibitors antipain, leupeptin, and soybean trypsin inhibitor (SBTI). TPA induced PA activity in normal 10T1/2 cells was inhibited by antipain. Transformed 10T1/2 cells maintained high levels of PA activity which were not further stimulated by the addition of TPA. Similarly, antipain inhibited the PA activity of the transformed cultures. Leupeptin and SBTI had no effect. These findings, in light of the fact that antipain has been shown to suppress the promotional effect of TPA in X-ray induced malignant transformation, may suggest a definite role for proteases in the transformational event or maintenance of the transformed state.

Topics: Animals; Antipain; Cell Transformation, Neoplastic; Cells, Cultured; Enzyme Induction; Female; Fibroblasts; Leupeptins; Mice; Oligopeptides; Phorbols; Plasminogen Activators; Plasminogen Inactivators; Pregnancy; Tetradecanoylphorbol Acetate; Time Factors; Trypsin Inhibitor, Bowman-Birk Soybean

We have investigated the effects of three protease inhibitors, antipain, leupeptin, and soybean trypsin inhibitor, on the induction of oncogenic transformation in mouse C3H10T 1/2 cells by X-rays. The patterns of inhibition by the three protease inhibitors were different. Antipain was the most effective, having the ability to suppress completely radiation transformation as well as radiation transformation enhanced by the phorbol ester promoting agent 12-O-tetradecanoylphorbol-13-acetate. The fact that antipain could suppress transformation when present for only 1 day following irradiation suggests that an effect on a DNA repair process might be important in its action. Leupeptin was less effective than antipain in its inhibition of radiation transformation. Soybean trypsin inhibitor suppressed only the promotional effects of 12-O-tetradecanoylphorbol-13-acetate on transformation. Our results suggest that there may be more than one protease involved in carcinogenesis.

Topics: Animals; Antipain; Cell Line; Cell Transformation, Neoplastic; DNA Repair; In Vitro Techniques; Leupeptins; Mice; Phorbol Esters; Protease Inhibitors; Radiation Effects; Time Factors

Topics: Animals; Antipain; Cell Survival; Cell Transformation, Neoplastic; Chromosome Aberrations; DNA Repair; Dose-Response Relationship, Radiation; In Vitro Techniques; Leupeptins; Mice; Phorbols; Protease Inhibitors; Sister Chromatid Exchange; Tetradecanoylphorbol Acetate; X-Rays

12-O-Tetradecanoylphorbol 13-acetate (TPA), a powerful tumor promoter, is shown to induce sister chromatid exchanges (SCEs), whereas the nonpromoting derivative 4-O-methyl-TPA does not. Inhibitors of tumor promotion--antipain, leupeptin, and fluocinolone acetonide--inhibit formation of such TPA-induced SCEs. TPA is a unique agent in its induction of SCEs in the absence of DNA damage, chromosome aberrations, mutagenesis, or significant toxicity. Because TPA is known to induce several gene functions, we speculate that it might also induce enzymes involved in genetic recombination. Thus, the irreversible step in tumor promotion might be the result of an aberrant mitotic segregation event leading to the expression of carcinogen/mutagen-induced recessive genetic or epigenetic chromosomal changes.

Topics: Cell Line; Cell Transformation, Neoplastic; Chromatids; Chromosome Aberrations; Chromosomes; Crossing Over, Genetic; Fluocinolone Acetonide; Leupeptins; Phorbols; Recombination, Genetic; Structure-Activity Relationship; Tetradecanoylphorbol Acetate

Topics: Animals; Anura; Cathepsins; Cell Adhesion; Cell Aggregation; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Concanavalin A; DNA, Neoplasm; Epithelium; In Vitro Techniques; Leupeptins; Male; Nitrosamines; Oligopeptides; Peptide Hydrolases; Rabbits; Rana catesbeiana; Urinary Bladder

The effect of six protease inhibitors, isolated from various species of actinomycetes, on focus formation by murine sarcoma virus was examined. Pepstatin was the only inhibitor. The treatment of cells with pepstatin at various times possibly retards the early stage of infection with murine sarcoma virus.

Topics: Animals; Carbamates; Cell Division; Cell Transformation, Neoplastic; Contact Inhibition; Dipeptides; Gammaretrovirus; Leupeptins; Mice; Mice, Inbred C57BL; Oligopeptides; Organophosphorus Compounds; Pancreatic Elastase; Pepstatins; Protease Inhibitors; Sarcoma Viruses, Murine; Thermolysin